This invention relates to a method of immobilizing microorganisms. More particularly, this invention is concerned with improvements in the fixing of microbial cells and enzymes on an insoluble support. The immobilized cells may then be used in any of a number of reactions, and the method has been found to be particularly useful for obtaining ethanol by fermentation processes, using immobilized viable cells.
Various techniques have been proposed for immobilizing microbial cells and enzymes on water-insoluble supports. Such methods have included covalent chemical linkage to a support via functional groups of the enzyme that are not essential to enzyme activity, entrapment or inclusion of the enzyme or cell within a hydrophilic gel lattice which retains the enzyme or cell but allows substrate and product to pass through, ionic binding or physical adsorption on hydrophilic ion exchanges or on charcoal or glass beads and cross-linking of the enzymes into large aggregates by reaction with bifunctional compounds.
However, ionic binding has not been uniformly acceptable because partial or total destruction of the enzyme or cell may result from a change in ionic strength, pH or temperature or addition of substrate when a totally insoluble preparation is derived.
Covalent linkage likewise has not provided an acceptable method in that the covalent linkages formed may be broken in use of the immobilized enzyme. Similarly, cross-linking of the enzymes has not been acceptable.
Although presenting the most versatility, entrapment or inclusion presents problems associated with the nature of the hydrophilic gel in which the enzyme or microbial cell is entrapped. That is, the hydrophilic support may be sufficient if used in monolayers, but not be able to withstand packing in a column. Additionally, it may be desirable to form uniform beads of support material with entrapped microbial cells or enzymes, and some supports may be incapable of being formed into beads or into any uniform shape without subjecting the support, with entrapped cells or enzymes, to extremity in temperature.
The immobilization of microbial cells and enzymes by inclusion or entrapment on hydrogels has long been known. However, only a few supports have been reported as successful for ethanol production, such as K-carrageenan and calcium alginate. The reason other supports have been acceptable for ethanol production is that the growth of cells and the production of carbon dioxide within the gel matrix weaken and rupture the gels. Although K-carrageenan and calcium alginate have reasonably high gel strengths which resist rupture, both present other problems. For example, K-carrageenan contains about 20% .lambda.-carrageenan. However, only K-carrageenan has gelling ability. The presence of .lambda.-carrageenan not only reduces the gel strength of the supports but also makes the carrageenan very viscous. Accordingly, crude carrageenan cannot be used for industrial application. Calcium alginate poses problems in that it is unstable in the presence of phosphate buffer and magnesium and potassium ions. Since phosphate salt is a major nutrient for microbial cells, the calcium alginate supports may not be appropriate for anything other than short-term experimentation.
Agar has also been used as a support. The advantage of agar is the fast growth of cells within the gel matrix. However, agar gel is brittle, and its gel strength is reduced by cell growth and the production of carbon dioxide during the initial stage of incubation. After incubation and rapid cell growth, the whole gel becomes very soft and brittle. Accordingly, agar is not suited for industrial applications.
Polyacrylamide has also been used as a support for entrapment of microbial cells and enzymes. Polyacrylamide, however, can only be formed into spherical beads of uniform size with great difficulty. Uniform, spherical bead-shape is a preferred shape.
Additionally, it has been found that ethanol production is drastically lower in those systems which employ polyacrylamide as a support. Accordingly, polyacrylamide gel as a support for entrapment of microbial cells and enzymes has severe use limitations.